Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum , produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)––by far the most abundant lysergic acid amide in M. brunneum ––are unknown. Ergot alkaloid synthesis ( eas ) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes ( easO and easP ) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella , indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine. Importance Fungi in the genus Metarhizium are important plant root symbionts and insect pathogens. They are formulated commercially to protect plants from insect pests. Several Metarhizium species including M. brunneum were recently shown to produce ergot alkaloids, a class of specialized metabolites studied extensively in other fungi because of their importance in agriculture and medicine. A biological role for ergot alkaloids in Metarhizium species had not been demonstrated previously. Moreover, the types of ergot alkaloids produced by Metarhizium species are lysergic acid amides, which have served directly or indirectly as important pharmaceutical compounds. The terminal steps in the synthesis of the most abundant lysergic acid amide in Metarhizium species and several other fungi (LAH) have not been determined. The results of this study demonstrate the role of a previously unstudied gene in LAH synthesis and indicate that LAH contributes to virulence of M. brunneum on insects.

Ergot alkaloids derived from lysergic acid have impacted humanity as contaminants of crops and as the bases of pharmaceuticals prescribed to treat dementia, migraines, and other disorders. Several plant-associated fungi in the Clavicipitaceae produce lysergic acid derivatives, but many of these fungi are difficult to culture and manipulate. Some Aspergillus species, which may be more ideal experimental and industrial organisms, contain an alternate branch of the ergot alkaloid pathway but none were known to produce lysergic acid derivatives. We mined genomes of Aspergillus species for ergot alkaloid synthesis ( eas ) gene clusters and discovered three species–– A. leporis, A. homomorphus, and A. hancockii ––had eas clusters indicative of the capacity to produce a lysergic acid amide. In culture, A. leporis, A. homomorphus, and A. hancockii produced lysergic acid amides, predominantly lysergic acid α-hydroxyethylamide (LAH). Aspergillus leporis and A. homomorphus produced high concentrations of LAH and secreted most of their ergot alkaloid yield into the culture medium. Phylogenetic analyses indicated genes encoding enzymes leading to the synthesis of lysergic acid were orthologous to those of the lysergic acid amide-producing Clavicipitaceae; however, genes to incorporate lysergic acid into an amide derivative evolved from different ancestral genes in the Aspergillus species. Our data demonstrate fungi outside the Clavicipitaceae produce lysergic acid amides and indicate the capacity to produce lysergic acid evolved once, but the ability to insert it into LAH evolved independently in Aspergillus species and the Clavicipitaceae. The LAH-producing Aspergillus species may be useful for study and production of these pharmaceutically important compounds. IMPORTANCE Lysergic acid derivatives are specialized metabolites with historical, agricultural, and medical significance and were known heretofore only from fungi in one family, the Clavicipitaceae. Our data show that several Aspergillus species, representing a different family of fungi, also produce lysergic acid derivatives and that the ability to put lysergic acid into its amide forms evolved independently in the two lineages of fungi. From microbiological and pharmaceutical perspectives, the Aspergillus species may represent better experimental and industrial organisms than the currently employed, lysergic acid producers of the plant-associated Clavicipitaceae. The observation that both lineages independently evolved the derivative lysergic acid α-hydroxyethylamide (LAH), among many possible lysergic acid amides, suggests a selection for this metabolite.

Objective The fungus Metarhizium brunneum produces ergot alkaloids of the lysergic acid amide class, most abundantly lysergic acid α-hydroxyethylamide (LAH). Genes for making ergot alkaloids are clustered in the genomes of producers. Gene clusters of LAH-producing fungi contain an α/β hydrolase fold protein-encoding gene named easP whose presence correlates with LAH production but whose contribution to LAH synthesis in unknown. We tested whether EasP contributes to LAH accumulation through gene knockout studies. Results We knocked out easP in M. brunneum via a CRISPR/Cas9-based approach, and accumulation of LAH was reduced to less than half the amount observed in the wild type. Because LAH accumulation was reduced and not eliminated, we identified and mutated the only close homolog of easP in the M. brunneum genome, a gene we named estA. An easP/estA double mutant did not differ from the easP mutant in lysergic acid amide accumulation, indicating estA had no role in the pathway. We conclude EasP contributes to LAH accumulation but is not absolutely required. Either a gene encoding redundant function and lacking sequence identity with easP resides outside the ergot alkaloid synthesis gene cluster, or EasP plays an accessory role in the synthesis of LAH.

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